The origin of neointimal cells in the rat carotid artery after balloon angioplasty

At present the issue of a possible role of circulating stem cells and precursors in pathological vascular wall remodeling after angioplasty remains unsolved. Therefore the origin of neointimal cells was examined in the rat carotid artery after balloon angioplasty using morphological and immunocytochemical approaches. It is shown that at the early stages (1-7 days) after vessel injury acute inflammatory response arises in the arterial wall recruiting neutrophils, monocytes, macrophages as well as large amounts of low-differentiated blood-derived cells. At the late stages (10-28 days), at the area of injured intima, a new hyperplastic intima (neointima) is formed, which consists of cells carrying specific smooth muscle markers--alpha-actin and smoothelin. The study on cell proliferative behaviour in the injured vessel wall by bromodeoxyuridine showed that in the process of neointima formation blood-born rather than resident cells are involved. Probably, early smooth muscle and endothelial precursor cells penetrate into injured area with blood stream, where they proliferative and differentiate into mature cells.     

Systemic alpha 1A-adrenoceptor antagonist inhibits neointimal growth after balloon injury of rat carotid artery

Previous in vitro and in vivo studies have shown that norepinephrine, acting through alpha(1A)-adrenoceptors, stimulates hypertrophy, proliferation, and migration of vascular smooth muscle cells and adventitial fibroblasts and may contribute to neointimal growth, lumen loss, and inward remodeling caused by iatrogenic wall injury and vascular disease. Our present aim was to determine whether intravenous administration of the alpha(1A)-adrenoceptor antagonist KMD-3213, at dosages without systemic hemodynamic effects, inhibits wall growth after injury. Inhibition of alpha(1A)-adrenoceptors with 12.8 and 32 microg/kg KMD-3213 had no effect on arterial pressure or renal and hindquarter resistances in anesthetized rats. A second group then received carotid balloon injury and continuous intravenous KMD-3213 at 4 and 10 microg x kg(-1) x h(-1) for 2 wk. Mean, systolic, and diastolic arterial pressures and heart rate of conscious unrestrained rats were unaffected. KMD-3213 reduced neointima growth by approximately 30 and 46% at the two doses (P < 0.01). These data support the novel hypothesis that a direct alpha(1A)-adrenoceptor-dependent trophic action of catecholamines is augmented by injury and may contribute significantly to hypertrophic vascular disease.     

Expression of interleukin 1 alpha and beta, and interleukin 1 receptor antagonist mRNA in the rat central nervous system after peripheral administration of lipopolysaccharides

Interleukin 1alpha (IL-1alpha) and IL-1beta, and the endogenous IL-1 receptor antagonist (IL-1ra) are known members of the IL-1 family. Using in situ hybridization histochemistry we demonstrated that following endotoxin injection (lipopolysaccharides, LPS, 2.0 mg/kg, i.p.) a time dependent expression and partly different expression patterns of the cytokines occurred within the rat brain and pituitary gland. All cytokines were observed in the choroid plexus. In addition, IL-1ra mRNA expressing cells were observed scattered in the brain parenchyma, whereas scattered IL-1beta mRNA expressing cells were restricted to central thalamic nuclei, the dorsal hypothalamus, and cortical regions, such as the parietal and frontal cortex. A strong IL-1beta mRNA expression was found in the circumventricular organs. In the pituitary gland, a low IL-1alpha and a high IL-1beta mRNA expression was observed, with the highest density of cytokine-expressing cells seen in the posterior pituitary. The cell types expressing the mRNA's of IL-1alpha, IL-1beta and IL-1ra were identified as monocytes in the circumventricular organs and the pituitary gland, and as microglia in the brain parenchyma. In conclusion, the present findings revealed that cytokine production in response to a peripheral endotoxin challenge mainly occurs in peripherally derived monocytes in the circumventricular organs and the pituitary gland. IL-1beta is the predominant form expressed, whereas the expression of IL-1alpha mRNA and IL-1ra mRNA is lower. Our observations support the view that peripherally derived IL-1 may play a role in the induction of centrally mediated illness symptoms.     

Expression of T-cadherin in the rat carotid artery wall after balloon injury and in different rat organs

T-cadherin is an unusual glycosilphosphatidylinositol (GPI)-anchored member of the cadherin family of cell adhesion proteins. In contrast to classical cadherins, tissue distribution of T-cadherin so far remained unknown. We examined tissue distribution of T-cadherin in rats using Western blotting and immunohistochemical method. Our results show that T-cadherin is expressed in all types of muscles (cardiac, striated, and smooth muscles), in brain neurons, and spinal cord, in the vessel endothelium, at the apical pole of intestinal villar epithelium, in the basal layer of skin, and eosophagal epithelium. Blood-derived and lymphoid cells as well as connective tissue were T-cadherin-negative. The highest level of T-cadherin expression was revealed in the cardiovascular system. Although T-cadherin was detected in smooth muscle cells, its role in the intimal thickening and restenosis is not known. We examined T-cadherin expression within 1-28 days after balloon injury of rat left carotid arteries. T-cadherin expression was valued immunohistochemically with semiquantitative method. In uninjured arteries, T-cadherin was expressed in endothelial (vWF-positive) cells, and smooth muscle (alpha-actin-positive) cells (SMCs). After denudation of arterial wall, T-cadherin was present both in the media and neointima. We revealed dynamics of T-cadherin expression in the media of injured artery: an essential increase being registered at the stage of cell migration and proliferation in the media and neointima (1-7 days), followed by its decrease to the baseline level (10-28 days). The high upregulation of T-cadherin expression in the media and neointima during migration and proliferation of vascular cells after vessel injury enables us to suggest the involvement of T-cadherin in vessel remodeling after balloon catheter injury.     

The interleukin-1 receptor antagonist influences interleukin-1 effects in rat and mouse

In this work we have focused on the ability of interleukin-1 to induce an acute phase protein response and a degranulation of polymorphonuclear leukocytes in vivo. The capacity of the interleukin-1 receptor antagonist to influence these events was also investigated. It was shown that interleukin-1 induced an acute phase protein response in rats and mice. In rats alpha(2)-macroglubolin levels were increased in plasma after an interleukin-1 injection whereas alpha(1)-inhibitor-3 decreased in plasma. In the mice plasma amyloid P was increased. The interleukin-1 receptor antagonist blocked the increase of alpha(2)-macroglobulin and plasma amyloid P in a dose dependent way while the effect on the alpha(1)-inhibitor-3 decrease was less pronounced. Interleukin-1 led to polymorphonuclear leukocyte degranulation in vivo as measured by increased cathepsin G plasma levels. The interleukin-1 receptor antagonist could influence the early phase of this degranulation.     

Nonselective ETA/ETB receptor antagonist blocks proliferation of rat vascular smooth muscle cells after balloon angioplasty

To elucidate the role of endothelin receptor subtypes in the abnormal proliferation of vascular smooth muscle cells (VSMC) associated with vascular injury, we have investigated the effects of a novel and potent nonselective ET_A/ET_B receptor antagonist (TAK-044) on the proliferation of rat VSMC in vitro and in vivo. TAK-044 dose-dependently inhibited DNA synthesis stimulated by 10⁻⁷ M ET-1 in cultured rat VSMC from the late passage with the approximate IC₅₀ of 6 × 10⁻⁸ M. After balloon angioplasty, the neointimal lesion in the injured carotid arteries in the TAK-044-treated group (0.052 ± 0.014 mm²) was significantly (p < 0.05) decreased compared to that in control group (0.26 ± 0.045 mm²), while the medial surface area was not affected. The intima/media ratio in the TAK-044 group (31 ± 6%) also significantly (p < 0.05) decreased from that of the control group (148 ± 25%). Our data suggest that nonselective ET_A/ET_B receptor antagonists may be therapeutic potential for prevention against the intimai thickening associated with vascular injury.     

重组人IL-1ra在大肠杆菌中的可溶性表达

利用PCR技术从含有IL-1ra的质粒上扩增IL-1ra基因,经过序列测定后插入表达载体pTIG-Trx,并转化大肠杆菌BL21(DE3),用IPTG进行诱导表达。经SDS-PAGE分析显示,IL-1ra表达质粒在大肠杆菌中的诱导表达产物出现相对分子量大约为17000的一条新生蛋白质带,其大小与预期结果一致,经Western和ELISA分析,证明该带即为目的蛋白,SDS-PAGE显示目的蛋白全部以可溶性蛋白的形式存在。超声破碎后,上清经金属螯和层析纯化获得纯度约为98％的蛋白样品。     

Human endometrial stromal interleukin-1 beta: autocrine secretion and inhibition by interleukin-1 receptor antagonist

Decidualization of human endometrial stromal cells is suppressed by endometrial IL-1 in an autocrine or paracrine manner, indicating that constant suppression of stromal decidualization by IL-1 requires a neutralizing mechanism for IL-1 action to accept embryo implantation. Since production of IL-1ra in human endometrium is reported to be 10- to 30-fold higher than that of IL-1 alpha/beta, we investigated whether endogenous IL-1 beta secreted from human endometrial stromal cells can be inhibited by IL-1ra by using an in vitro decidualization culture. Human stromal cells were cultured with 8-Br-cAMP to induce decidualization, and concentrations of IL-1 beta, IL-8, and prolactin in the culture supernatants were assayed before and after decidualization. There was no significant difference in mean IL-1 beta concentrations measured before and after decidualization. Addition of IL-1ra to endometrial stromal cell cultures strongly inhibited endogenous IL-8 secretion from the cells. Although IL-1 beta showed a biphasic effect on cell proliferation and a suppressive effect on decidualization of stromal cells, these effects were completely inhibited by IL-1ra. The results imply that a high in vivo concentration of IL-1ra in human endometrial tissues may regulate IL-1 effects on decidualization and cell proliferation of human endometrial stromal cells.     

Expression of interleukin-1 beta and interleukin-1 receptor antagonist in oxLDL-treated human aortic smooth muscle cells and in the neointima of cholesterol-fed endothelia-denuded rabbits

The migration of vascular smooth muscle cells (VSMCs) from the media to the intima and the proliferation of intimal VSMCs are key events in restenotic lesion development. These events, which are preceded and accompanied by inflammation, are modulated by the proinflammatory cytokine, interleukin-1 beta (IL-1 beta), which induces vascular smooth muscle cells to express adhesion molecules and to proliferate. IL-1 beta action is complex and regulated, in part, by its naturally occurring inhibitor, the IL-1 receptor antagonist (IL-1ra). Whether there was a temporal and spatial correlation between IL-1 beta and IL-1ra expression in, and release by, oxidized low density lipoproteins (oxLDL)-stimulated human aortic smooth muscle cells (HASMCs) was determined by using ELISA and Western blot. In addition, IL-1 beta and IL-1ra expression was detected in the neointima of endothelia-denuded cholesterol-fed New Zealand white rabbits by immunohistochemistry and Western blot. In HASMCs, oxLDL induced IL-beta and IL-1ra expression and release in a dose- and time-dependent manner. Treatment with 20 microg/ml oxLDL resulted in increased IL-1 beta release after 6 h, which peaked at 24 h, and in increased IL-1ra release, first seen after 12 h, but continuing to increase for at least 48 h. In the cells, IL-beta expression showed a similar pattern to release, whereas IL-1ra expression was seen in unstimulated cells and was not increased by oxLDL treatment. Confocal microscopy showed colocalization of IL-beta and IL-1ra expression in oxLDL-stimulated HASMCs. oxLDL caused significant induction of nuclear factor kappa B and activator protein-1 DNA binding activity in HASMCs (6.6- and 3.3-fold, respectively). In cholesterol-fed endothelia-denuded rabbits, the notably thickened intima showed significant IL-1 beta and IL-1ra expression. These results provide further support for the role of IL-1 system in the pathogenesis of restenosis. This is the first demonstration of IL-1 beta and IL-1ra expression and secretion of oxLDL-treated HASMCs and their expression in the rabbit neointima, suggesting that the smooth muscle cells of the intima are an important source of these factors.     

Expression and characterization of human glycosylated interleukin-1 receptor antagonist in Pichia pastoris

Interleukin-1 receptor antagonist is an inhibitor of the pro-inflammatory action of interleukin-1. The gene encoding for interleukin-1 receptor antagonist (IL-1ra) was cloned into a Pichia pastoris expression vector pPICzalphaA (Invitrogen, USA) and transformed into P. pastoris strain SMD1168H. Multi-copy selection of the gene produced a high expressing strain of IL-1ra that produced 17mg/L of total secreted purified protein. The IL-1ra produced in P. pastoris was a mixture of glycosylated and non-glycosylated IL-1ra where 70% of the total protein was glycosylated. SP-Sepharose purification allowed for separation of the two expressed forms of IL-1ra, which permits biochemical investigation of glycosylated and non-glycosylated IL-1ra using one expression system. Mass spectrometric analysis revealed the expression of the full-length protein and that the glycosylated IL-1ra contained high mannose glycoforms that ranged from Man(9)GlcNAc(2) to Man(14)GlcNAc(2).     

Novel interleukin-1 receptor antagonist exon polymorphisms and their use in allele-specific mRNA assessment

A variable number of tandem repeats (VNTR) polymorphism has been described in intron 2 of the interleukin-1 receptor antagonist gene. Allele 2 of this polymorphism is associated with many chronic inflammatory diseases. Using direct sequencing of polymerase chain reaction products from individuals of known genotype for the VNTR, we have identified four single base change polymorphisms in exons 1_ic and 2 and one upstream of exon 1_ic, all of which are probably in linkage disequilibrium with the intron 2 VNTR. The exonic polymorphisms do not alter the encoded amino acid sequence. Using the exon 2 polymorphism as a marker for the intron 2 disease-associated allele, we have been able to analyse allele-specific mRNA in heterozygotic keratinocyte cell lines. The disease-associated allele shows no difference from other alleles in this cell type with respect to mRNA accumulation. Received: 17 July 1995 / Revised: 4 December 1995     

Altered interleukin-1 receptor antagonist and interleukin-18 mRNA expression in myocardial tissues of patients with dilatated cardiomyopathy

Interleukin-1 (IL-1) is a potent regulator of cell proliferation, inflammation, and contraction of cardiovascular cells. It has been proposed that the IL-1/IL-1ra (IL-1 receptor antagonist) ratio influences these functions. Other members of the IL-1 family and the related caspase-1 also contribute to regulation of IL-1-mediated functions. We determined the mRNA expression of caspase-1, caspase-3, IL-1alpha , IL-1beta , IL-18, IL-1 receptor type I (IL-1-RI), and IL-1ra in left ventricle tissue of hearts from patients with ischemic or dilated cardiomyopathy (ICM or DCM) and in control tissues from unused donor transplant hearts in RT-PCR experiments. We show that the expression of caspase-1, caspase-3, IL-1beta , and IL-1-RI mRNA was not different between patients and control tissues. Furthermore, we did not find detectable amounts of IL-1alpha mRNA in any of these adult myocardial tissues. On the other hand, expression of IL-18 RNA was lower in myocardium of both patient groups compared with control hearts. Furthermore, IL-1ra mRNA expression was significantly lower in tissues of DCM patients compared with ICM patients and controls. This was in line with a trend towards lower IL-1ra protein levels in myocardial tissues of DCM patients. In contrast with the adult tissues discussed above, which did not express IL-1alpha mRNA, commercially available human fetal tissue expressed IL-1alpha mRNA. On the other hand IL-1beta mRNA was present in fetal and in adult human heart tissue. Our data provide evidence for an altered ratio of IL-1/IL-1ra in DCM patients. This dysregulation may contribute to pathogenesis and/or progression of heart disease by modulating the otherwise balanced IL-1-mediated functions in cardiovascular cells.     

Induction of gicerin/CD146 in the rat carotid artery after balloon injury

Gicerin is a cell adhesion molecule belonging to the immunoglobulin superfamily. It is reported that the human homologous molecule, CD146, is expressed in the endothelial cells. Here, we found that the expression of gicerin was increased in the rat carotid arteries after balloon injury. Immunohistochemical analysis demonstrated that the expression of gicerin protein was increased in the medial smooth muscle cells prior to the formation of neointima one week after the injury and was also increased in the luminal edge of the neointima after two weeks. We employed A10 cells, a cell line derived from rat aortic smooth muscle cell, and examined the effect of growth factors on the expression of gicerin, such as IGF-1, PDGF-BB, and bFGF. We found that IGF-1, but not PDGF-BB and bFGF, significantly increases the expression of gicerin protein in A10 cells. These suggest gicerin might be involved in the arteriosclerotic neointima formation in the artery.     

Over expression of interleukin-1alpha,interleukin-1beta and interleukin-1 receptor antagonist in testicular tissues from sexually immature mice as compared to adult mice

The levels of IL-1alpha, IL-1beta and IL-1Ra were higher in homogenates of testicular tissue from sexually immature than those from mature mice. Immunohistochemical staining of testicular tissues from sexually immature and adult mice show that differentiated germ cells express higher levels of IL-1alpha compared to Sertoli cells and Leydig cells/interstitial cells. Peritubular cells of sexually immature and adult mice did not express IL-1alpha. Testicular tissue cells of adult mice showed high levels of expression of IL-1beta, mainly in the cytoplasm and nucleus of the spermatogonia and in spermatocytes. Sertoli cells and Leydig/interstitial cells were also highly stained for IL-1beta. However, peritubular cells did not express IL-1beta. On the other hand, testicular tissue cells from sexually immature mice, showed high levels of IL-1beta, mainly in spermatocytes. Spermatogonia showed low levels of IL-1beta expression. Also, high levels of IL-1beta expression were detected in Leydig/interstitial cells. Peritubular cells clearly showed IL-1beta expression. Testicular tissue cells from adult mice, showed IL-1Ra expression in spermatogonia, Sertoli and Leydig/interstitial cells. IL-1Ra expression was clearly present in the Golgi apparatus of spermatogonia and Sertoli cells. However, peritubular cells did not show IL-1Ra expression. Testicular tissue cells from sexually immature mice, also showed high levels of IL-1Ra expression mainly in the cytoplasm and nucleus of the spermatogonia and Sertoli cells. In addition, Leydig/interstitial cells and peritubular cells also expressed IL-1Ra. Our results demonstrate, for the first time, the expression of IL-1beta in germ and Sertoli cells, and IL-1Ra in Leydig/interstitial cells of testicular tissues from adult and sexually immature mice, under in vivo conditions. In addition, the relative elevated levels of the IL-1 system in the testis of immature mice compared to mature mice may indicate its involvement in the spermatogenesis.     

Inhibition of endothelin-converting enzyme for protection against neointimal proliferation following balloon angioplasty of the rat carotid artery

Clinical success of percutaneous transluminal coronary angioplasty is limited by restenosis within months of the initial intervention. A number of vasoactive mediators and growth factors have been reported to participate in this process. The aim of the present experiments was to examine the effects of nonselective neutral endopeptidase (NEPi)/endothelin-converting enzyme (ECEi) inhibitors against neointimal proliferation (NIP) following balloon angioplasty of the left carotid artery of Sprague-Dawley rats with the right vessel serving as the uninjured control. The rats were divided in several groups: group 1, nontreated (vehicle); group 2, treated with a selective NEPi i.p.; groups 3-7, treated with nonselective NEPi/ECEi either i.p., s.c., i.v., or p.o. at various doses. After 2 weeks, cross-sectional histopathological and morphometrical examination of the left carotids revealed a severe NIP in vehicle-treated angioplastic rats compared with the control uninjured right carotid of the same rats. The selective NEPi CGS 24592 had no significant effect on restenosis, nor did the dual NEPi/ECEi CGS 26303 at 5 mg x kg(-1) x day(-1) i.p. Both s.c and i.v. NEPi/ECEi treatment (10 mg x kg(-1) x day(-1) b.i.d. s.c. or 10 mg x kg(-1) x day(-1) i.v.) reduced NIP by up to 35%. The prodrug CGS 26393 (p.o.) also attenuated NIP by 23%. Plasma concentrations of these compounds correlated with the degree of inhibition. These data support the participation of the endothelin system in the rat model of balloon angioplasty and suggest that selective ECEi may be effective.     

Identification of a high-affinity receptor for interleukin-1 beta in rat brain

A single type of high-affinity binding sites for IL-1 beta was identified in the rat hypothalamus (Kd = 1.0 +/- 0.2 nM) and cerebral cortex (Kd = 1.3 +/- 0.2 nM), but not in the pituitary. The maximum binding capacity (Bmax) in the hypothalamus (Bmax = 75.4 +/- 10.8 fmol/mg protein) was 4 times greater than in the cerebral cortex (Bmax = 17.2 +/- 1.5 fmol/mg protein). Neither various neuropeptides nor IL-2 appeared to influence the binding of [125I]IL-1 beta to the hypothalamic membrane preparations. The potency of unlabeled IL-1 alpha to replace the binding of [125I]IL-1 beta to the hypothalamic membrane preparations was considerably less than that of unlabeled IL-1 beta. These findings indicate that IL-1 beta receptors are heterogeneously distributed in the central nervous system and that IL-1 alpha does not bind with IL-1 beta receptors in the brain.     

Physiologic role of interleukin-1 receptor antagonist

Recent studies have described the spontaneous development of arthritis or vasculitis in IL-1 receptor antagonist (IL-1Ra) knockout mice bred on specific and different genetic backgrounds. The levels of both secreted and intracellular isoforms of IL-1Ra produced in the rheumatoid joint or in the arterial wall may not be adequate to effectively inhibit the excess amounts of locally produced IL-1. Thus, an imbalance between IL-1 and IL-1Ra may predispose to local inflammatory disease in particular tissues in the presence of other as yet unknown genetically influenced factors.